There are five major types of dopamine receptors, classified into two primary categories, D1-like and D2-like. The D1-like family consists of the D1 and D5 dopamine receptors, whereas, D2-like family consists of the D2, D3, and D4 dopamine receptors. The D1 dopamine receptor (D1R), in particular, may be linked to a variety of neuropsychiatric disorders and represents an attractive drug target for the enhancement of cognition. Agents that enhance D1R signaling in the prefrontal cortex may be useful in the treatment of cognitive decline in schizophrenia, Alzheimer's disease and other age-related disorders. In FY17, we continued characterizing two D1 positive allosteric modulator (PAM) scaffolds, MLS1082 and MLS6585, previously identified in a high throughput screen. MLS1082 and MLS6585 potentiate dopamine (DA)-stimulated G-protein and beta-arrestin-mediated D1R signaling, increasing the potency of DA for stimulating cAMP accumulation and for beta-arrestin recruitment. Both compounds also increased the maximum DA-stimulated responses. Further, the two compounds potentiated the affinity of DA to bind to the D1R. Importantly, they showed no ability to potentiate forskolin-stimulated cAMP accumulation, or beta-arrestin recruitment by the D2-like DA receptors. Experiments using maximally effective concentrations of MLS1082 and MLS6585 in combination were used to determine if the compounds were acting at separate or similar binding locations. The combination of MLS1082 + MLS6585 was additive in potentiating DA's potency for stimulating beta-arrestin recruitment and cAMP accumulation, suggesting the two compounds are acting at separate sites. Repeating these combination experiments with Compound B, a known D1R PAM, showed additive activity with only MLS6585 and not MLS1082, further suggesting that there are separate PAM binding sites. We are currently engaged in a chemical optimization campaign to synthesize analogs around these scaffolds to both interrogate structure-activity relationships and optimize compounds for in vivo testing. Despite its clinical importance in the treatment of a number of neuropsychiatric disorders, such as Parkinson's disease and schizophrenia, there are few compounds that are highly selective for the D2 DA receptor (D2R). Most compounds with activity at the D2R also exhibit significant affinity for the D3 or D4 DA receptors (D3R or D4R), or other G protein-coupled receptors (GPCRs). In FY17, we continued optimizing selective antagonists for the D2R that we previously identified in a high-throughput screen. One compound, MLS6916 is 700-fold selective for the D2R vs. D3R as determined in functional and radioligand binding assays. MLS6916 was examined for functional activity at 168 different GPCRs and showed no agonist activity on any GPCR tested, and only exhibited antagonist activity at the D2-like receptors. Preliminary investigations, however, revealed that this initial hit compound was metabolically unstable. To optimize this scaffold, we conducted an iterative medicinal chemistry campaign with the goals of increasing selectivity, potency, and engendering metabolic stability. Analogs were analyzed for activity on D2R, D3R, and D4R using both radioligand binding and functional assays to determine affinities and potencies for the D2-like receptors. In addition, analogs were tested for solubility, permeability, and stability. Several lead compounds were identified with Ki values of <50 nM for the D2R with >1,000-fold selectivity vs. the D3R. These selectivity trends were confirmed in functional beta-arrestin recruitment assays. Importantly, lead compounds with enhanced metabolic stability and permeability were identified. Further analysis and optimization of these lead compounds will provide new insight into highly selective D2R antagonists for the treatment of DA-related disorders. The D2 DA receptor (D2R) signals through a variety of second messenger pathways making it challenging to discern which are linked to specific effects of D2R-targeted drugs; however, this complexity provides a unique opportunity to develop pathway-selective therapeutics. Our laboratory previously described MLS1547 as a functionally selective D2R ligand that robustly activates G-protein signaling with minimal recruitment of beta-arrestin. Structure-activity analyses of a series of analogs with varying bias, coupled with molecular dynamics, led to a molecular model for biased signaling including a hydrophobic binding pocket formed by residues I184, F189, and V190, all located within the fifth transmembrane region (TM5) of the D2R. In FY17, we used mutagenesis techniques to test this model and investigate the role of TM5 in regulating D2R signaling bias. We constructed single point mutations (I184A, V190A, F189A, F189L, and F189Y) in the D2R and studied their G protein-mediated signaling and beta-arrestin recruitment using BRET and enzyme complementation-based technologies. While there was a minimal change in potency, mutation of either I184 or V190 showed no effect on DA's efficacy for beta-arrestin recruitment or activation of G-protein-mediated signaling. Interestingly, when we examined the F189A mutant, the ability of DA and other D2R agonists to recruit beta-arrestin was lost, while the G protein-signaling efficacy was maintained. DA-stimulated beta-arrestin recruitment was also lost in the F189L mutant, but maintained in the more conserved F189Y mutant receptor. G-protein activation was unaffected by the F189L or F189Y mutations. These data demonstrate that the F189A and the F189L D2R mutants are biased towards G protein-mediated signaling and suggest that the F189 residue is important for stabilizing an activated state for recruiting beta-arrestin. These results further suggest that conformational changes in TM5 can act as a molecular switch for receptor signaling via beta-arrestin recruitment, which may have implications for the design of novel biased compounds for the treatment of D2R-related disorders The D3R is enriched in the striatum, which is associated with control of movement, mood and emotion. In an effort to discover highly selective compounds for the D3R, our lab employed a high throughput screen of the NIH small molecule library. Hits were counter screened against the D2R to allow for the elucidation of compounds that activate the D3R without effects on the D2R. Orthogonal confirmation and counter-screens were also performed to obtain an initial assessment of selectivity and mechanisms of action. The most promising compound was chosen for a full optimization study and investigation of its structure-activity relationships. 375 analogs were synthesized and screened in the beta-arrestin assay in an effort to increase both affinity and selectivity. The lead compound identified through this process, ML417, acts as a full agonist at the D3R with a potency of 36 nM, while having minimal effects on D2R-mediated beta-arrestin translocation. ML417 also acts as a potent and selective D3R agonist in a separate BRET-based assay of beta-arrestin recruitment. Importantly, the compound also exhibits potent and selective agonist activity in D3R-G protein-mediated signaling responses as demonstrated using Go-BRET-based assays and pERK assays. ML417 was further assessed for receptor cross-reactivity using panels of other GPCRs, and was found to have limited liability for either functional interactions with, or displacement of a radioligand from numerous GPCRs. As D3R-preferring orthosteric agonists show promise as neuroprotective and neurorestorative agents, we conducted preliminary studies using ML417 in a neuroprotective assay, and found that it displays neuroprotective properties. This highly selective and potent D3R agonist will prove useful as a research tool and may show utility as a therapeutic drug lead.